Little is known about the pathogenesis of Lyme Disease and current diagnostic tests are inadequate. In an effort to utilize molecular methodologies to address these problems, we recently cloned a library of Borrelia burgdorferi strain 297 DNA. One clone of chromosomal DNA, Ly-1, is specific for B. burgdorferi (Bb), hybridizing with all strains studied, and does not cross-hybridize with other DNAs. The Ly-I clone has been used to sensitively detect Bb by dot-blot hybridization, and, in tissues of infected hamsters, by in situ hybridization. We have produced Bb specific oligonucleotide primers from the Ly-I sequence and used these in the polymerase chain reaction (PCR) to detect <10 copies of Bb DNA and, in preliminary studies, to detect Bb in urine in both early and late Lyme Disease (LD) in humans. We now propose using Ly-I in molecular pathogenic and diagnostic studies of experimental, veterinary, and human LD. Experimentally infected hamsters and their offspring will be studied, using PCR and DNA hybridization, to determine the course and duration of infection and specific tissue and organ tropisms, the effects of prior immunity and whether treatment truly eradicates infection. The PCR will be used on human and veterinary urine, plasma and other body fluid samples obtained in our endemic area to determine its diagnostic sensitivity and specificity in various stages of disease. In both suspected LD and in setting (e.g. canine nephritis) where Bb has a possible, but unproven, role, pathological specimens (for example skin, synovium, kidney, placenta) will be examined by PCR and in situ hybridization for Bb DNA. These studies should allow the accurate determination of 1) the distribution and localization of the spirochete in infected tissues, 2) the role of active infection and/or persistence in late disease and treatment failures 3) the clinical spectrum of veterinary and human LD and 4) the potentially important role of molecular hybridization and the PCR as diagnostic tests. Taken together, this new information should be extremely useful to clinicians and laboratory investigators in their ongoing efforts to better define, understand, prevent and treat Lyme borreliosis.